The invention is an adjunct for enhancing the performance of immunoassays for detecting the presence of analyte in serum. The invention relates to methods and kits for pretreating serum so as to dissociate the analyte from the serum antibody in preparation for an immunoassay analysis of the analyte. More particularly, the invention relates to the use of chaotropic acids for dissociating analyte from serum antibody.
Immunoassays are often employed for detecting the presence of analyte. An example of one such immunoassay is disclosed by David et al. U.S. Pat. No. 4,376,110), commonly known as a "sandwich" immunoassay. According to David's immunoassay, analyte is first exposed to and bound to an immobilized antibody having a specificity for the analyte; the presence of such bound analyte is then determined by a second binding by a labeled soluable antibody, also having a specificity for the analyte. The labeled soluable antibody is conjugated to a label which is easily detected. The label is often an enzyme. After the unbound component of the labeled antibody is washed away, the labeled antibody which remains is proportional to the amount of analyte originally exposed to the immobilized antibody. The immunoassay is called a "sandwich" immunoassay because the analyte is sandwiched between two antibodies, viz. the immobilized antibody and the soluble antibody conjugated to a label.
The immunoassay described by David and similar ELISA procedures may be employed for detecting analyte in serum. However, the performance of such immunoassays will be impaired if the serum sample includes serum antibody having a specificity for the particular analyte. If the analyte is antigenic with respect to the serum donor, the serum sample may include antibody generated by a donor against such analyte. If such serum antibody is not removed from the serum prior to the immunoassay, it will interfere with the performance of commonly employed immunoassays, such as the David immunoassay. The degree of such interference is determined by the donor's antibody titer and the avidity of such serum antibody. In turn, the donor's antibody titer will depend upon the antigenicity of the analyte and the course of the donor's immune response to such analyte.
William F. Feller et al. (European Patent Application Ser. No. 85400002.3: "Brest Cancer Diagnostic Blood Test") disclose adjunct methods for improving the performance of immunoassays for the analysis of serum or plasma analytes. The Feller method employs either chaotropic salts or an aqueous dilution procedure to dissociate the serum of plasma analyte from serum antibody. The dissociated analyte is then dried within the well of a microtiter plate, coating the walls thereof. If chaotropic salts are employed, the residual salts may then be washed from the well. During such wash, the analyte remains attached to the walls of the well and then becomes the object of a subsequent immunoassay procedure.
F. K. de Steenwinkel et al. (European Patent Application Ser. No. 81301559.1: "Agglutination Immunoassays") discloses the addition of chaotropic agents to particle agglutination immunoassays for dissociating analyte from nonspecifically bound serum proteins. Free analyte causes specific agglutination between antibody treated particles. The chaotropic agent remains present throughout the particle agglutination immunoassay. In order to avoid interference with the agglutination process, the concentration of added chaotrope is carefully controlled. If too little chaotrope is present, the nonspecific interference is unabated; if too much chaotrope is present, the specific agglutination is prevented. The de Steenwinkel reference does not disclose that the dissociation and agglutination phases may be separated; the de Steenwinkel reference does not disclose the use of halogenated chaotropic acids.
S. S. Stone et al. (Journal of Immunological Methods, vol. 31, pp 379-387 (1979): "Isolation and Purification of Bovine IgM by Dissociating Immunoglobulin-Brucella Complexes") discloses and compares the dissociation of antigen-antibody complexes by the use of water extraction, chaotropic salts (various concentrations of KSCN), and low pH (2M glycine at pH 2.0). The antibody yield for Stone's pH treatment was poor compared to the water extraction and chaotropic salt treatments (9.2% or less). However, subsequent work has shown that the application of denaturing heat (65.degree. C.) with a pH procedure such as Stone's significantly enhances the dissociation process and the resultant yield.
William H. Sawyer (The Journal of Biological Chemistry, vol. 248 (24), pp. 8429-8433 (1973): "The Dissociation of Proteins by Chaotropic Salts") discloses and compares the use of chaotropic salts, including the sodium salts of certain halogenated chaotropic acids, buffered between pH 4.6 and pH 7.5, with temperatures between 4.degree. C. and 14.degree. C., to dissociate various proteins. Sawyer found that the effectiveness of the tested chaotropes for dissociating these proteins was partially predicted by the Hofmeister series. Although Sawyer's study included the sodium salts of halogenated chaotropic acids, the study did not determine the effectiveness of halogenated chaotropes as free acids. Between pH 4.6 and pH 7.5, each of the chaotropes studied by Sawyer exist as ionic salts, having lost their dissociable proton.
H. von Schenck et al. (Scandinavian Journal of Laboratory Investigation, vol. 43, pp. 527-531 (1983): "Ligand Leakage from Immunoaffinity Column") discloses the use of 1-2M aqueous solutions of formic acid, as a chaotropic agent, for eluting antigen-antibody complexes from immunoaffinity columns. Formic acid is sometimes favored for eluting bound antigen from immunoaffinity columns because, after the elution, the formic acid may then be separated from the eluant by lyophylization. However, von Schenck does not represent that formic acid may be similarly employed for dissociating antigen-antibody complexes within a serum sample; nor does von Schenck disclose the use of halogenated chaotropic acids for dissociating antigen-antibody complexes.